Progress in FY2009 was in the following areas: (1) QUANTITATIVE DETERMINATION OF SITE-SPECIFIC CONFORMATIONAL DISTRIBUTIONS IN AN UNFOLDED PROTEIN: In work that has been published recently in the Journal of Molecular Biology, we carried out solid state NMR measurements that directly probe backbone phi and psi torsion angles at three specific sites in the model protein villin HP35, one site in each of the three helical segments of the folded state. These measurements employ three techniques developed previously in our group, with abbreviated names CT-DQFD, DQCSA, and 2DEXMAS. When these techniques were applied to HP35 in its folded state (0.0 M GdnHCl), the data were fit best by a single conformation, very close to the alpha-helical phi and psi angles determined by crystallography for folded HP35. In the partially unfolded state (4.5 M GdnHCl), the combined data could not be fit by a single conformation. Instead, the data were well described by significant populations of helical and extended, beta-strand-like conformations. In the fully unfolded state (7.0 M GdnHCl), the combined data were best fit by a mixture of extended and polyproline II conformations, with no significant population of alpha-helical conformations. Thus, these measurements define an "unfolding pathway", in which helical segments first convert to extended conformations, and finally to polyproline II conformations. Site-specific variations in the population ratios of the two components were observed. (2) RAPID FREEZE-QUENCHING OF AN INTERMEDIATE STATE IN PROTEIN FOLDING: We have constructed an apparatus that permits rapid freezing of protein solutions from high temperatures, in approximately 10 microseconds. With this apparatus, we have carried out the first solid state NMR studies of a transient intermediate state in a protein folding process. Again using HP35, we find that rapid freezing from +90 C (above the thermal unfolding temperature) to -130 C (in cold isopentane liquid) traps an unexpected state of the protein, in which "unfolded" molecules coexist with "folded" molecules, but in which the "folded" molecules are in fact not fully folded. Instead, the experimental 1D and 2D solid state NMR data indicate that the "folded" component has native-like secondary structure but incomplete formation of tertiary structure. Full folding involves a slower phase of structural relaxation, perhaps on the 0.1-1.0 ms time scale, in which sidechain packing is optimized. These results have implications for the interpretation of other types of experimental measurements on fast-folding proteins, and for attempts by theorists to fold small proteins such as HP35 in computational studies.